Particle‐by‐Particle In Situ Characterization of the Protein Corona via Real‐Time 3D Single‐Particle‐Tracking Spectroscopy**

Tan, Xiaochen; Welsher, Kevin

doi:10.1002/ange.202105741

Cited by 8 publications

(9 citation statements)

References 58 publications

(106 reference statements)

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“…More detailed studies are required to characterize the type of protein−nanoparticles interactions, such as protein layer density or protein geometry. Specifically in situ single particle binding kinetics studies, 20 for which holoNTA is ideally suited, offer a promising route to obtain this information.…”

Section: Resultsmentioning

confidence: 99%

“…Solutions to the issue of finite track lengths have been explored in the form of high speed acquisitions, 18 volume confinement via nanofluidic channels, 19 or lock-on detection. 20 However, in the absence of specialized hardware, holographic sensing-platforms with immobilized particles are often the only option for sizing small NPs. These methods are able to detect and quantify particles as small as single proteins in a label-free fashion, but estimate size purely based on scattering amplitudes (σ S ) or particle-induced phase-changes.…”

mentioning

confidence: 99%

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Simultaneous Sizing and Refractive Index Analysis of Heterogeneous Nanoparticle Suspensions

et al. 2022

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Rapid and reliable characterization of heterogeneous nanoparticle suspensions is a key technology across the nanosciences. Although approaches exist for homogeneous samples, they are often unsuitable for polydisperse suspensions, as particles of different sizes and compositions can lead to indistinguishable signals at the detector. Here, we introduce holographic nanoparticle tracking analysis, holoNTA, as a straightforward methodology that decouples size and material refractive index contributions. HoloNTA is applicable to any heterogeneous nanoparticle sample and has the sensitivity to measure the intrinsic heterogeneity of the sample. Specifically, we combined high dynamic range k-space imaging with holographic 3D single-particle tracking. This strategy enables long-term tracking by extending the imaging volume and delivers precise and accurate estimates of both scattering amplitude and diffusion coefficient of individual nanoparticles, from which particle refractive index and hydrodynamic size are determined. We specifically demonstrate, by simulations and experiments, that irrespective of localization uncertainty and size, the sizing sensitivity is improved as our extended detection volume yields considerably longer particle trajectories than previously reported by comparable technologies. As validation, we measured both homogeneous and heterogeneous suspensions of nanoparticles in the 40−250 nm size range and further monitored protein corona formation, where we identified subtle differences between the nanoparticle−protein complexes derived from avidin, bovine serum albumin, and streptavidin. We foresee that our approach will find many applications of both fundamental and applied nature where routine quantification and sizing of nanoparticles are required.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Simultaneous Sizing and Refractive Index Analysis of Heterogeneous Nanoparticle Suspensions

et al. 2022

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“…49 Isolating the chemical growth rates from the physical requires performing these same experiments in a good solvent�that is, to obtain the resolution of the chemical growth rates of single polymer strands in solution. While a good solvent will dramatically increase the diffusional speed of the growing polymers (by enabling measurement of smaller, single strands), a combination of increased solvent viscosity, photon-efficient scan patterns, 59 and algorithms for extracting small signals in large backgrounds 60 should make the tracking of the growth of single polymer chains in solution possible via 3D-SMART in future studies. As a substantial portion of all chemical processes occur in the solution phase, these studies enable a wealth of nonensemble averaged chemical insights into synthetically important systems.…”

Section: ■ Conclusionmentioning

confidence: 99%

Growth Kinetics of Single Polymer Particles in Solution via Active-Feedback 3D Tracking

García

Blum

et al. 2022

J. Am. Chem. Soc.

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The ability to directly observe chemical reactions at the single-molecule and single-particle level has enabled the discovery of behaviors otherwise obscured by ensemble averaging in bulk measurements. However powerful, a common restriction of these studies to date has been the absolute requirement to surface tether or otherwise immobilize the chemical reagent/reaction of interest. This constraint arose from a fundamental limitation of conventional microscopy techniques, which could not track molecules or particles rapidly diffusing in three dimensions, as occurs in solution. However, many chemical processes occur entirely in the solution phase, leaving single-particle/-molecule analysis of this critical area of science beyond the scope of available technology. Here, we report the first kinetics studies of freely diffusing and actively growing single polymer−particles at the singleparticle level freely diffusing in solution. Active-feedback single-particle tracking was used to capture three-dimensional (3D) trajectories and real-time volumetric images of freely diffusing polymer particles (D ≈ 10 −12 m 2 /s) and extract the growth rates of individual particles in the solution phase. The observed growth rates show that the average growth rate is a poor representation of the true underlying variability in polymer−particle growth behavior. These data revealed statistically significant populations of fasterand slower-growing particles at different depths in the sample, showing emergent heterogeneity while particles are still freely diffusing in solution. These results go against the prevailing premise that chemical processes in freely diffusing solution will exhibit uniform kinetics. We anticipate that these studies will launch new directions of solution-phase, nonensemble-averaged measurements of chemical processes.

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“…This makes RT-3D-SPT a powerful tool to investigate fast single-particle and single-molecule phenomena, such as cellular trafficking, 9,10 nanoparticle-membrane interactions, 11,12 DNA transcription, 13–15 and countless other dynamic processes. 16–18…”

Section: Introductionmentioning

confidence: 99%

Active-feedback 3D single-molecule tracking using a fast-responding galvo scanning mirror

Tan

Hou

Zhang

et al. 2023

Preprint

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Real-time three-dimensional single-particle tracking (RT-3D-SPT) allows continuous detection of individual freely diffusing objects with high spatiotemporal precision by applying closed-loop active feedback in an optical microscope. However, the current tracking speed in RT-3D-SPT is primarily limited by the response time of control actuators, impeding long-term observation of fast diffusive objects such as single molecules. Here, we present an RT-3D-SPT system with improved tracking performance by replacing the XY piezoelectric stage with a galvo scanning mirror with an approximately five-time faster response rate (~5 kHz). Based on the previously developed 3D single-molecule active real-time tracking (3D-SMART), this new implementation with a fast-responding galvo mirror eliminates the mechanical movement of the sample and allows more rapid response to particle motion. The improved tracking performance of the galvo mirror-based implementation is verified through simulation and proof-of-principle experiments. Fluorescent nanoparticles and ~ 1 kB double-stranded DNA molecules were tracked via both the original piezoelectric stage and new galvo mirror implementations. With the new galvo-based implementation, notable increases in tracking duration, localization precision, and the degree to which the objects are locked to the center of the detection volume were observed. These results suggest faster control response elements can expand RT-3D-SPT to a broader range of chemical and biological systems.

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Particle‐by‐Particle In Situ Characterization of the Protein Corona via Real‐Time 3D Single‐Particle‐Tracking Spectroscopy**

Cited by 8 publications

References 58 publications

Simultaneous Sizing and Refractive Index Analysis of Heterogeneous Nanoparticle Suspensions

Simultaneous Sizing and Refractive Index Analysis of Heterogeneous Nanoparticle Suspensions

Growth Kinetics of Single Polymer Particles in Solution via Active-Feedback 3D Tracking

Active-feedback 3D single-molecule tracking using a fast-responding galvo scanning mirror

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